- Email: [email protected]
Mutations in BRAT1 cause autosomal recessive progressive encephalopathy: Report of a Spanish patient
Accepted Manuscript Mutations in BRAT1 Cause Autosomal Recessive Progressive Encephalopathy: Report of a Spanish Patient Dr. Alberto Fernández-Jaén, Sara Álvarez, Eui Young So, Toru Ouchi, Mar Jiménez de la Peña, Anna Duat, Daniel Martín Fernández-Mayoralas, Ana Laura FernándezPerrone, Jacobo Albert, Beatriz Calleja-Pérez PII:
S1090-3798(16)00042-8
DOI:
10.1016/j.ejpn.2016.02.009
Reference:
YEJPN 2018
To appear in:
European Journal of Paediatric Neurology
Received Date: 13 September 2015 Revised Date:
19 December 2015
Accepted Date: 14 February 2016
Please cite this article as: Fernández-Jaén A, Álvarez S, So EY, Ouchi T, Jiménez de la Peña M, Duat A, Fernández-Mayoralas DM, Fernández-Perrone AL, Albert J, Calleja-Pérez B, Mutations in BRAT1 Cause Autosomal Recessive Progressive Encephalopathy: Report of a Spanish Patient, European Journal of Paediatric Neurology (2016), doi: 10.1016/j.ejpn.2016.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fernández-Jaén et al. 1
ACCEPTED MANUSCRIPT
Mutations in BRAT1 Cause Autosomal Recessive Progressive Encephalopathy: Report of a Spanish Patient.
Short Title: BRAT1 and Progressive Encephalopathy.
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Authors: Alberto Fernández-Jaéna, Sara Álvarezb, Eui Young Soc, Toru Ouchic, Mar Jiménez de la Peñad, Anna Duate, Daniel Martín Fernández-Mayoralasa, Ana Laura Fernández-Perronea, Jacobo Albertf, Beatriz Calleja-Pérezg
b
c
SC
Department of Pediatric Neurology, Hospital Universitario Quirón. Madrid. Spain. Genomics and Medicine, NIMGenetics. Madrid. Spain.
Department of Cancer Genetics, Roswell Park Cancer Institute. Buffalo. New York.
M AN U
a
USA. d
Neuro-Radiology Unit, Magnetic Resonance Unit, Hospital Universitario Quirón.
Madrid. Spain. e
f
TE D
Pediatric Neurology Unit, Hospital Universitario Niño Jesús. Madrid. Spain.
Department of Biological and Health Psychology. Faculty of Psychology. Universidad
Autónoma de Madrid. g
EP
Pediatric Primary Care, C. S. Doctor Cirajas. Madrid. Spain.
Communication should be addressed to:
AC C
Dr. Alberto Fernandez Jaén.
Hospital Universitario Quirón Madrid. Neurology Service. Head of Pediatric Neurology. C/ Diego de Velazquez 1, 28024 Pozuelo de Alarcón, Madrid. Spain. E-mail: [email protected] Work phone: +34 902151016 Fax number: +34 915183232
Fernández-Jaén et al. 2
ACCEPTED MANUSCRIPT ABSTRACT
We describe a 4-year-old male child born to non-consanguineous Spanish parents with progressive encephalopathy (PE), microcephaly, and hypertonia. Whole exome sequencing revealed compound heterozygous BRAT1 mutations [c.1564G>A (p.Glu522Lys) and c.638dup (p.Val214Glyfs*189)]. Homozygous and compound
rigidity and multifocal seizure syndrome (MIM# 614498).
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heterozygous BRAT1 mutations have been described in patients with lethal neonatal The seven previously
described patients suffered from uncontrolled seizures, and all of those patients died in their first months of life. BRAT1 acts as a regulator of cellular proliferation and
SC
migration and is required for mitochondrial function. The loss of these functions may explain the cerebral atrophy observed in this case of PE. This case highlights the extraordinary potential of next generation technologies for the diagnosis of rare genetic
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diseases, including PE. Making a prompt diagnosis of PE is important for genetic counselling and disease management. Key Words:
Progressive encephalopathy, whole exome sequencing, BRAT1, lethal neonatal
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EP
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rigidity.
Fernández-Jaén et al. 3
ACCEPTED MANUSCRIPT
INTRODUCTION Progressive encephalopathy (PE) is rarely seen in general pediatric practice. Children may present with unexplained psychomotor retardation and signs of
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progressive CNS diseases. The incidence of PE is unknown but has been estimated to be 0.5-0.6 per 1,000 live births (1, 2). Stromme et al. [2007] calculated an overall incidence rate for PE of 6.43 per 100,000 person years in Oslo, Norway using local population data (1). PE is
SC
predominantly caused by inborn errors of metabolism or neurodegenerative diseases without an identifiable metabolic deficiency (1, 3). Importantly, the risk of PE and other
of known consanguinity (4-6).
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disabilities increases in consanguineous unions or in communities with a high incidence These data support the predominantly autosomal
recessive inheritance pattern for described causes of PE.
In addition, mortality rates are increased significantly in patients with PE in comparison to the total population. Patient fatality varies from 17 to 37% (3, 7); this rate
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is higher in patients with neonatal onset and/or underlying metabolic diseases (7). Recently, homozygous or compound heterozygous BRAT1 mutations have been described as a new cause of severe PE with neonatal onset and high patient fatality (811). The seven earlier described cases suffered from refractory epilepsy, and all of them
EP
died in their first months of life. We describe a new patient with compound heterozygous BRAT1 mutations and PE but with some atypical characteristics; he is
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alive at the age of 4.5 years and he never presented seizures. CASE REPORT
This 4-year-old male patient was the first child of nonconsanguineous parents of
Spanish origin. He was the product of a 38-week-pregnancy and born via uncomplicated C-section to a young primigravida mother. Caesarean was selected due to a podalic presentation. The Apgar scores were 9 and 9 at 1 and 5 minutes, respectively. The birth weight was 2,700 gr (5th percentile), the length was 47 cm (5th percentile), and the OFD was 34 cm (25th percentile).
Fernández-Jaén et al. 4
ACCEPTED MANUSCRIPT
The patient was evaluated by us during the fourth year of life due to psychomotor retardation and progressive microcephaly. At that age, his weight was 14 kg (5th percentile), his height was 100 cm (30th percentile), and his OFD was 47 cm (<3rd percentile) (Fig. 1). Severe axial hypotonia, exaggerated deep-tendon reflexes and Babinski reflex were also observed. No abnormal movements or dysmorphic features
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were perceived during the physical examination. An early intervention program was established in his first months of life, including motor, cognitive, speech development and social behavior approaches. In spite of this therapy, the patient exhibited a severe psychomotor delay during his first
SC
years of life. Head control was observed at 4 months, he sat unsupported at 30 months, he was able to stand with help at 42 months, and a social smile was observed at 12 months. Although the first bisyllabic babbling occurred at 12 months, language
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development was limited to some unmeaning sounds at the age of 4 years. His severe psychomotor retardation made impossible an appropiate cognitive evaluation. The results of routine laboratory screening, including thyroid function, and neurometabolic tests were within normal range. Lactic and pyruvic acid were normal in serum and cerebrospinal fluid. Mitochondrial respiratory chain enzymatic activities on
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muscle tissue were normal at the age of 3 years old. Sleep and sleep-deprived videoEEG tests performed at the ages of 1, 2 and 4 years old, displayed normal results (Figure 1). The eye and ear examinations yielded normal results. In addition, both visual and auditory evoked potentials were normal at the age of 3 years old.
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Conventional genetic studies (karyotype and array comparative genomic hybridization analysis) revealed no abnormalities. Two timed/spaced out 1.5T MRI of the brain, which was performed at the ages
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of 19 and 48 months, showed moderate progressive cerebellar atrophy (Fig. 2-3). Whole exome sequencing revealed two mutations in the BRAT1 gene (BRCA1-
Associated ATM Activator 1) at chromosome 7 positions g.2579211C>T, c.1564G>A; p.Glu522Lys and g. 2583388C>CT, c.638dupA, p.Val214Glyfs*189 [NM_152743]. Compound heterozygosity of the identified mutations was confirmed, as both variants were identified in heterozygosity in the mother and father, consistent with autosomal recessive inheritance. The BRAT1 p.Glu522Lys mutation occurred at a highly conserved amino acid in the HEAT (this acronym comes from four cytoplasmic proteins: Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the yeast kinase TOR1)
Fernández-Jaén et al. 5
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protein domain. Many HEAT repeat-containing proteins have been noted to be involved in intracellular transport processes. The BRAT1 p.Glu522Lys mutation was predicted to be damaging by sorting intolerant from tolerant (SIFT), PolyPhen2, MutationTaster, provean and LRT. The second variant, BRAT1 p.Val214Serfs*189, was predicted to change the amino acid sequence after valine 214 by producing a stop codon that
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prematurely truncates the protein 188 positions downstream. An allelic frequency of 0.005% has been reported by the exome aggregation consortium in the European (nonFinnish) population (12). However, the pathogenicity of this variant has been reported previously, as this variant has been identified as a homozygous frameshift mutation
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associated with lethal neonatal rigidity and multifocal seizure syndrome (MIM# 614498) in 3 patients (8).
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METHOD: GENETIC STUDY
Exome sequencing was performed using genomic DNA isolated (MagnaPure, Roche) from whole blood from the proband and his parents. Libraries were prepared using the Ion AmpliSeq™ Exome Kit (Life Technologies) and quantified by qPCR. The enriched libraries were prepared using Ion Chef™ and sequenced on a PI™ Chip in the
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Ion Proton™ System (Life Technologies) to provide at least 20X coverage of >90% of amplicons. Signal processing, base calling, alignment and variant calling were performed on a Proton™ Torrent Server using the Torrent Suite™ Software. Variants were annotated using Ion Reporter™ Software, and pedigree analysis was performed
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using the Genetic Disease Screen (GDS) trio workflow. Variant filtering and prioritization were performed using an in-house software program and a local database.
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Candidate variants were visualized using IGV (Integrative Genomics Viewer). Candidate variants were evaluated based on stringent assessments at both the gene and variant levels, taking into consideration both the patient’s phenotype and the inheritance pattern. Variants were classified according to the guidelines of the American College of Medical Genetics and Genomics (ACMG). A board-certified molecular clinical geneticist evaluated each variant and classified it as pathogenic, likely pathogenic, or variant of uncertain significance. The geneticist then decided which, if any, variants should be reported. In every case, causal variants were discussed with the referring physician and/or a clinical geneticist. Identified variants were confirmed by Sanger sequencing.
Fernández-Jaén et al. 6
ACCEPTED MANUSCRIPT DISCUSSION
We describe an infrequent case of PE caused by compound heterozygous BRAT1 mutations with some distinct manifestations. Previously reported patients had died in the first months of life, and all of those patients suffered from severe intractable seizures (Table I). The first report described two unrelated Amish infants with lethal,
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multifocal seizures, hypertonia, microcephaly, apnea, and bradycardia (8). After this report, five other cases were described. Saunders et al. [2012] described a new case from Mexico with lethal, seizures, microcephaly, dysmorphic features, congenital heart defects, and severe dyspnea; the presence of several tracts of homozygosity in aCGH
SC
analysis suggested shared parentage (10). Japanese siblings with compound heterozygous mutations in BRAT1 were described recently; those siblings showed some
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dysmorphic features, arrested head growth, uncontrolled seizures, and hypertonia. Brain MRI showed cerebral and cerebellar atrophy in both cases (9). One new report described two siblings born to consanguineous Arab-Muslim parents who have mutations in the same gene, with myoclonic seizures, progressive microcephaly, hypertonia, and apnea-bradycardia (11). Our case also had microcephaly and hypertonia; however, he was still alive at the age of 4.5 years and no seizures were
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observed by parents, therapists or clinicians; no paroxysmal events were recorded during the sleep and sleep-deprived video-EEG either. The BRAT1 gene encodes the BRCA1-associated ATM activation-1 protein
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(BRAT1) (13). This protein interacts with the BRCT domain of the tumor suppressor gene BRCA1 and binds to ATM1 and DNA-PKs, suggesting a role of BRAT1 in DNA damage responses (13-15). BRAT1 is also involved in cellular growth processes,
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including cell proliferation and tumorigenicity, and required for mitochondrial functions (16). The loss of BRAT1 expression due to homozygous or compound heterozygous BRAT1 mutations may decrease cell proliferation and migration, affect mitochondrial homeostasis (16), and cause neuronal atrophy (8, 10). The lower severity observed in our patient might be related to his genotype or the absence of epilepsy. Aggressive ictal and electrical epileptogenic activity during brain maturation are believed to contribute to progressive cognitive deterioration or regression (17). Genomic advances have transformed clinical practice, and these cases demonstrate that whole exome sequencing (WES) has the potential to change clinical
Fernández-Jaén et al. 7
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practice for genetic diseases. Making a correct and prompt diagnosis is important for genetic counselling, prenatal diagnostic options, carrier testing, and the prevention of disease. In some genetic disorders, the identification of causative mutations may have the potential to lead to new therapies or to change the management of acutely ill infants (10, 18).
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In summary, we describe an infrequent case of PE, hypertonia, progressive microcephaly, and severe psychomotor retardation with compound heterozygous BRAT1 mutations. Our case demonstrates that this gene is related to PE, even without epilepsy. Until now, scientific references have probably reported the most severe cases.
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As other colleagues have suggested, BRAT1 should be added to the list of genes that are related to PE (11). Finally, we should insist on the necessity of genetic studies in
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patients with PE. Additional investigations will be worthwhile to determine the prevalence and contribution of these genes mutations to PE. Despite the clinical and etiology diversity of PE, genetic studies may be decisive for genetic counselling and
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management.
Fernández-Jaén et al. 8
ACCEPTED MANUSCRIPT REFERENCES
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1. Stromme P, Kanavin OJ, Abdelnoor M, et al. Incidence rates of progressive childhood encephalopathy in Oslo, Norway: a population based study. BMC pediatrics. 2007;7:25. PubMed PMID: 17597517. Pubmed Central PMCID: 1914055. 2. Surtees R. Understanding neurodegenerative disorders. Curr Paediatr. 2002;12(13):191-8. 3. Tomas Vila M, Vitoria Minana I, Gomez Gosalvez F, et al. [Epidemiology of progressive intellectual and neurological deterioration in childhood. A multicentre study in the Community of Valencia]. Anales de pediatria. 2013 May;78(5):303-7. PubMed PMID: 23063114. Epidemiologia del deterioro intelectual y neurologico progresivo en la infancia. Estudio multicentrico en la Comunidad Valenciana. 4. Nelson J, Smith M, Bittles AH. Consanguineous marriage and its clinical consequences in migrants to Australia. Clinical genetics. 1997 Sep;52(3):142-6. PubMed PMID: 9377802. 5. Weller M, Tanieri M, Pereira JC, et al. Consanguineous unions and the burden of disability: a population-based study in communities of Northeastern Brazil. American journal of human biology : the official journal of the Human Biology Council. 2012 Nov-Dec;24(6):835-40. PubMed PMID: 23042425. 6. Stromme P, Suren P, Kanavin OJ, et al. Parental consanguinity is associated with a seven-fold increased risk of progressive encephalopathy: a cohort study from Oslo, Norway. European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society. 2010 Mar;14(2):138-45. PubMed PMID: 19446480. 7. Stromme P, Magnus P, Kanavin OJ, et al. Mortality in childhood progressive encephalopathy from 1985 to 2004 in Oslo, Norway: a population-based study. Acta paediatrica. 2008 Jan;97(1):35-40. PubMed PMID: 18076719. 8. Puffenberger EG, Jinks RN, Sougnez C, et al. Genetic mapping and exome sequencing identify variants associated with five novel diseases. PloS one. 2012;7(1):e28936. PubMed PMID: 22279524. Pubmed Central PMCID: 3260153. 9. Saitsu H, Yamashita S, Tanaka Y, et al. Compound heterozygous BRAT1 mutations cause familial Ohtahara syndrome with hypertonia and microcephaly. Journal of human genetics. 2014 Dec;59(12):687-90. PubMed PMID: 25319849. 10. Saunders CJ, Miller NA, Soden SE, et al. Rapid whole-genome sequencing for genetic disease diagnosis in neonatal intensive care units. Science translational medicine. 2012 Oct 3;4(154):154ra35. PubMed PMID: 23035047. Pubmed Central PMCID: 4283791. 11. Straussberg R, Ganelin-Cohen E, Goldberg-Stern H, et al. Lethal neonatal rigidity and multifocal seizure syndrome--report of another family with a BRAT1 mutation. European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society. 2015 Mar;19(2):240-2. PubMed PMID: 25500575. 12. Exome Aggregation Consortium ExAC. ExAC Browser Beta 2015 [updated July]. Available from: http://exac.broadinstitute.org. 13. Aglipay JA, Martin SA, Tawara H, Lee SW, Ouchi T. ATM activation by ionizing radiation requires BRCA1-associated BAAT1. The Journal of biological chemistry. 2006 Apr 7;281(14):9710-8. PubMed PMID: 16452482. 14. Ouchi M, Ouchi T. Regulation of ATM/DNA-PKcs Phosphorylation by BRCA1-Associated BAAT1. Genes & cancer. 2010 Dec;1(12):1211-4. PubMed PMID: 21779444. Pubmed Central PMCID: 3092282. 15. So EY, Ouchi T. Functional interaction of BRCA1/ATM-associated BAAT1 with the DNAPK catalytic subunit. Experimental and therapeutic medicine. 2011 May;2(3):443-7. PubMed PMID: 22977523. Pubmed Central PMCID: 3440717.
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16. So EY, Ouchi T. BRAT1 deficiency causes increased glucose metabolism and mitochondrial malfunction. BMC cancer. 2014;14:548. PubMed PMID: 25070371. Pubmed Central PMCID: 4129107. 17. Khan S, Al Baradie R. Epileptic encephalopathies: an overview. Epilepsy research and treatment. 2012;2012:403592. PubMed PMID: 23213494. Pubmed Central PMCID: 3508533. 18. Willig LK, Petrikin JE, Smith LD, et al. Whole-genome sequencing for identification of Mendelian disorders in critically ill infants: a retrospective analysis of diagnostic and clinical findings. The Lancet Respiratory medicine. 2015 May;3(5):377-87. PubMed PMID: 25937001. Pubmed Central PMCID: 4479194.
Fernández-Jaén et al. 10
ACCEPTED MANUSCRIPT FIGURE LEGENDS
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Figure 1.
Sleep video-EEG test, performed on a 25-month-old boy. Normal EEG results; no
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EP
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epileptic paroxysms were registered.
Fernández-Jaén et al. 11
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Figures 2-3.
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On the left side, MRI study performed on a 19-month-old boy (2a-2b). 2a. Midsagittal T1-weighted image exhibits a severe vermian diffuse atrophy (arrow) with fourth ventricle enlargement. The corpus callosum is normal, and the brainstem
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has a normal size.
2b. Axial T2-weighted image shows enlarged interfolia spaces (arrow) and moderately
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reduced volume of both cerebellar hemispheres. On the right side, MRI study performed at 48 months old (3a-3b). 3a. Midsagittal T1-weighted image evidences a more severe vermian diffuse atrophy (arrow) with enlargement of the supravermian cistern and a wide fourth ventricle. The corpus callosum and the brainstem remain normal. 3b. Axial T2-weighted image shows severe symmetric atrophy of the cerebellar hemispheres (arrow).
Fernández-Jaén et al. 12
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Table I. Clinical features, brain MRI findings and BRAT1 mutations. Puffenberg
Saunders CJ, et al.
Saitsu H
Straussbe
EG, et al.
(10)
et al. (9)
rg R, et
N=1
N=2
N=2
N=2
Clinical
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Features Dysmorphic
-
+
+
-
-
+ (at birth)
+ (at birth)
+
+
+
(postnatal
(postnatal
(postnatal
)
)
)
+
+
+
+
+
+
-
+
-
+
-
-
+
-
-
-
+
-
+
Rigidity/Hyper +
+
+
Dysautonomia
+
(apnea, bradycardia)
EP
Seizures
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tonia
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features Microcephaly
N=1
al. (11)
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(8)
Our case
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Brain MRI Cerebral
+ (mild
atrophy
hypoplasia of frontal lobes)
Cerebellar atrophy
-
Fernández-Jaén et al. 13
ACCEPTED MANUSCRIPT Died
Before age
Age 5 months
4 months
Ages 1
Before
Alive at
year and
age 6
52
9 months
months
months
(1st case) and 3
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months (2nd case) Homozygo
mutation
us
Homozygous c.453_454insATCT
c.638_639i
TCTC
Homozyg
Compoun
d
ous
d
heterozyg ous
c.1173del G
heterozyg ous
c.176T>C
c.1564G>
and
A and
c.962_96
c.638dup
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nsA
Compoun
SC
BRAT1
3del
Amish
Mexican
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EP
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Population
Japanese Siblings
ArabMuslim Siblings
Spanish
Fernández-Jaén et al. 14
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Acknowledgements: Part of this work was supported by R01CA79892, R01CA90631,
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EP
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SC
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and 5P30CA16056 from the National Institutes of Health, USA (T.O.).
ACCEPTED MANUSCRIPT Highlights • • •
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•
Homozygous and compound heterozygous BRAT1 mutations are related to severe progressive encephalopathy. The loss of BRAT1 expression may decrease neuronal proliferation and migration. We describe the oldest patient with BRAT1 mutations, progressive encephalopathy and without seizures. Whole exome sequencing may be useful for identifying the causes of progressive encephalopathies.
S1090-3798(16)00042-8
DOI:
10.1016/j.ejpn.2016.02.009
Reference:
YEJPN 2018
To appear in:
European Journal of Paediatric Neurology
Received Date: 13 September 2015 Revised Date:
19 December 2015
Accepted Date: 14 February 2016
Please cite this article as: Fernández-Jaén A, Álvarez S, So EY, Ouchi T, Jiménez de la Peña M, Duat A, Fernández-Mayoralas DM, Fernández-Perrone AL, Albert J, Calleja-Pérez B, Mutations in BRAT1 Cause Autosomal Recessive Progressive Encephalopathy: Report of a Spanish Patient, European Journal of Paediatric Neurology (2016), doi: 10.1016/j.ejpn.2016.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fernández-Jaén et al. 1
ACCEPTED MANUSCRIPT
Mutations in BRAT1 Cause Autosomal Recessive Progressive Encephalopathy: Report of a Spanish Patient.
Short Title: BRAT1 and Progressive Encephalopathy.
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Authors: Alberto Fernández-Jaéna, Sara Álvarezb, Eui Young Soc, Toru Ouchic, Mar Jiménez de la Peñad, Anna Duate, Daniel Martín Fernández-Mayoralasa, Ana Laura Fernández-Perronea, Jacobo Albertf, Beatriz Calleja-Pérezg
b
c
SC
Department of Pediatric Neurology, Hospital Universitario Quirón. Madrid. Spain. Genomics and Medicine, NIMGenetics. Madrid. Spain.
Department of Cancer Genetics, Roswell Park Cancer Institute. Buffalo. New York.
M AN U
a
USA. d
Neuro-Radiology Unit, Magnetic Resonance Unit, Hospital Universitario Quirón.
Madrid. Spain. e
f
TE D
Pediatric Neurology Unit, Hospital Universitario Niño Jesús. Madrid. Spain.
Department of Biological and Health Psychology. Faculty of Psychology. Universidad
Autónoma de Madrid. g
EP
Pediatric Primary Care, C. S. Doctor Cirajas. Madrid. Spain.
Communication should be addressed to:
AC C
Dr. Alberto Fernandez Jaén.
Hospital Universitario Quirón Madrid. Neurology Service. Head of Pediatric Neurology. C/ Diego de Velazquez 1, 28024 Pozuelo de Alarcón, Madrid. Spain. E-mail: [email protected] Work phone: +34 902151016 Fax number: +34 915183232
Fernández-Jaén et al. 2
ACCEPTED MANUSCRIPT ABSTRACT
We describe a 4-year-old male child born to non-consanguineous Spanish parents with progressive encephalopathy (PE), microcephaly, and hypertonia. Whole exome sequencing revealed compound heterozygous BRAT1 mutations [c.1564G>A (p.Glu522Lys) and c.638dup (p.Val214Glyfs*189)]. Homozygous and compound
rigidity and multifocal seizure syndrome (MIM# 614498).
RI PT
heterozygous BRAT1 mutations have been described in patients with lethal neonatal The seven previously
described patients suffered from uncontrolled seizures, and all of those patients died in their first months of life. BRAT1 acts as a regulator of cellular proliferation and
SC
migration and is required for mitochondrial function. The loss of these functions may explain the cerebral atrophy observed in this case of PE. This case highlights the extraordinary potential of next generation technologies for the diagnosis of rare genetic
M AN U
diseases, including PE. Making a prompt diagnosis of PE is important for genetic counselling and disease management. Key Words:
Progressive encephalopathy, whole exome sequencing, BRAT1, lethal neonatal
AC C
EP
TE D
rigidity.
Fernández-Jaén et al. 3
ACCEPTED MANUSCRIPT
INTRODUCTION Progressive encephalopathy (PE) is rarely seen in general pediatric practice. Children may present with unexplained psychomotor retardation and signs of
RI PT
progressive CNS diseases. The incidence of PE is unknown but has been estimated to be 0.5-0.6 per 1,000 live births (1, 2). Stromme et al. [2007] calculated an overall incidence rate for PE of 6.43 per 100,000 person years in Oslo, Norway using local population data (1). PE is
SC
predominantly caused by inborn errors of metabolism or neurodegenerative diseases without an identifiable metabolic deficiency (1, 3). Importantly, the risk of PE and other
of known consanguinity (4-6).
M AN U
disabilities increases in consanguineous unions or in communities with a high incidence These data support the predominantly autosomal
recessive inheritance pattern for described causes of PE.
In addition, mortality rates are increased significantly in patients with PE in comparison to the total population. Patient fatality varies from 17 to 37% (3, 7); this rate
TE D
is higher in patients with neonatal onset and/or underlying metabolic diseases (7). Recently, homozygous or compound heterozygous BRAT1 mutations have been described as a new cause of severe PE with neonatal onset and high patient fatality (811). The seven earlier described cases suffered from refractory epilepsy, and all of them
EP
died in their first months of life. We describe a new patient with compound heterozygous BRAT1 mutations and PE but with some atypical characteristics; he is
AC C
alive at the age of 4.5 years and he never presented seizures. CASE REPORT
This 4-year-old male patient was the first child of nonconsanguineous parents of
Spanish origin. He was the product of a 38-week-pregnancy and born via uncomplicated C-section to a young primigravida mother. Caesarean was selected due to a podalic presentation. The Apgar scores were 9 and 9 at 1 and 5 minutes, respectively. The birth weight was 2,700 gr (5th percentile), the length was 47 cm (5th percentile), and the OFD was 34 cm (25th percentile).
Fernández-Jaén et al. 4
ACCEPTED MANUSCRIPT
The patient was evaluated by us during the fourth year of life due to psychomotor retardation and progressive microcephaly. At that age, his weight was 14 kg (5th percentile), his height was 100 cm (30th percentile), and his OFD was 47 cm (<3rd percentile) (Fig. 1). Severe axial hypotonia, exaggerated deep-tendon reflexes and Babinski reflex were also observed. No abnormal movements or dysmorphic features
RI PT
were perceived during the physical examination. An early intervention program was established in his first months of life, including motor, cognitive, speech development and social behavior approaches. In spite of this therapy, the patient exhibited a severe psychomotor delay during his first
SC
years of life. Head control was observed at 4 months, he sat unsupported at 30 months, he was able to stand with help at 42 months, and a social smile was observed at 12 months. Although the first bisyllabic babbling occurred at 12 months, language
M AN U
development was limited to some unmeaning sounds at the age of 4 years. His severe psychomotor retardation made impossible an appropiate cognitive evaluation. The results of routine laboratory screening, including thyroid function, and neurometabolic tests were within normal range. Lactic and pyruvic acid were normal in serum and cerebrospinal fluid. Mitochondrial respiratory chain enzymatic activities on
TE D
muscle tissue were normal at the age of 3 years old. Sleep and sleep-deprived videoEEG tests performed at the ages of 1, 2 and 4 years old, displayed normal results (Figure 1). The eye and ear examinations yielded normal results. In addition, both visual and auditory evoked potentials were normal at the age of 3 years old.
EP
Conventional genetic studies (karyotype and array comparative genomic hybridization analysis) revealed no abnormalities. Two timed/spaced out 1.5T MRI of the brain, which was performed at the ages
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of 19 and 48 months, showed moderate progressive cerebellar atrophy (Fig. 2-3). Whole exome sequencing revealed two mutations in the BRAT1 gene (BRCA1-
Associated ATM Activator 1) at chromosome 7 positions g.2579211C>T, c.1564G>A; p.Glu522Lys and g. 2583388C>CT, c.638dupA, p.Val214Glyfs*189 [NM_152743]. Compound heterozygosity of the identified mutations was confirmed, as both variants were identified in heterozygosity in the mother and father, consistent with autosomal recessive inheritance. The BRAT1 p.Glu522Lys mutation occurred at a highly conserved amino acid in the HEAT (this acronym comes from four cytoplasmic proteins: Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the yeast kinase TOR1)
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protein domain. Many HEAT repeat-containing proteins have been noted to be involved in intracellular transport processes. The BRAT1 p.Glu522Lys mutation was predicted to be damaging by sorting intolerant from tolerant (SIFT), PolyPhen2, MutationTaster, provean and LRT. The second variant, BRAT1 p.Val214Serfs*189, was predicted to change the amino acid sequence after valine 214 by producing a stop codon that
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prematurely truncates the protein 188 positions downstream. An allelic frequency of 0.005% has been reported by the exome aggregation consortium in the European (nonFinnish) population (12). However, the pathogenicity of this variant has been reported previously, as this variant has been identified as a homozygous frameshift mutation
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associated with lethal neonatal rigidity and multifocal seizure syndrome (MIM# 614498) in 3 patients (8).
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METHOD: GENETIC STUDY
Exome sequencing was performed using genomic DNA isolated (MagnaPure, Roche) from whole blood from the proband and his parents. Libraries were prepared using the Ion AmpliSeq™ Exome Kit (Life Technologies) and quantified by qPCR. The enriched libraries were prepared using Ion Chef™ and sequenced on a PI™ Chip in the
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Ion Proton™ System (Life Technologies) to provide at least 20X coverage of >90% of amplicons. Signal processing, base calling, alignment and variant calling were performed on a Proton™ Torrent Server using the Torrent Suite™ Software. Variants were annotated using Ion Reporter™ Software, and pedigree analysis was performed
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using the Genetic Disease Screen (GDS) trio workflow. Variant filtering and prioritization were performed using an in-house software program and a local database.
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Candidate variants were visualized using IGV (Integrative Genomics Viewer). Candidate variants were evaluated based on stringent assessments at both the gene and variant levels, taking into consideration both the patient’s phenotype and the inheritance pattern. Variants were classified according to the guidelines of the American College of Medical Genetics and Genomics (ACMG). A board-certified molecular clinical geneticist evaluated each variant and classified it as pathogenic, likely pathogenic, or variant of uncertain significance. The geneticist then decided which, if any, variants should be reported. In every case, causal variants were discussed with the referring physician and/or a clinical geneticist. Identified variants were confirmed by Sanger sequencing.
Fernández-Jaén et al. 6
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We describe an infrequent case of PE caused by compound heterozygous BRAT1 mutations with some distinct manifestations. Previously reported patients had died in the first months of life, and all of those patients suffered from severe intractable seizures (Table I). The first report described two unrelated Amish infants with lethal,
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multifocal seizures, hypertonia, microcephaly, apnea, and bradycardia (8). After this report, five other cases were described. Saunders et al. [2012] described a new case from Mexico with lethal, seizures, microcephaly, dysmorphic features, congenital heart defects, and severe dyspnea; the presence of several tracts of homozygosity in aCGH
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analysis suggested shared parentage (10). Japanese siblings with compound heterozygous mutations in BRAT1 were described recently; those siblings showed some
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dysmorphic features, arrested head growth, uncontrolled seizures, and hypertonia. Brain MRI showed cerebral and cerebellar atrophy in both cases (9). One new report described two siblings born to consanguineous Arab-Muslim parents who have mutations in the same gene, with myoclonic seizures, progressive microcephaly, hypertonia, and apnea-bradycardia (11). Our case also had microcephaly and hypertonia; however, he was still alive at the age of 4.5 years and no seizures were
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observed by parents, therapists or clinicians; no paroxysmal events were recorded during the sleep and sleep-deprived video-EEG either. The BRAT1 gene encodes the BRCA1-associated ATM activation-1 protein
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(BRAT1) (13). This protein interacts with the BRCT domain of the tumor suppressor gene BRCA1 and binds to ATM1 and DNA-PKs, suggesting a role of BRAT1 in DNA damage responses (13-15). BRAT1 is also involved in cellular growth processes,
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including cell proliferation and tumorigenicity, and required for mitochondrial functions (16). The loss of BRAT1 expression due to homozygous or compound heterozygous BRAT1 mutations may decrease cell proliferation and migration, affect mitochondrial homeostasis (16), and cause neuronal atrophy (8, 10). The lower severity observed in our patient might be related to his genotype or the absence of epilepsy. Aggressive ictal and electrical epileptogenic activity during brain maturation are believed to contribute to progressive cognitive deterioration or regression (17). Genomic advances have transformed clinical practice, and these cases demonstrate that whole exome sequencing (WES) has the potential to change clinical
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practice for genetic diseases. Making a correct and prompt diagnosis is important for genetic counselling, prenatal diagnostic options, carrier testing, and the prevention of disease. In some genetic disorders, the identification of causative mutations may have the potential to lead to new therapies or to change the management of acutely ill infants (10, 18).
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In summary, we describe an infrequent case of PE, hypertonia, progressive microcephaly, and severe psychomotor retardation with compound heterozygous BRAT1 mutations. Our case demonstrates that this gene is related to PE, even without epilepsy. Until now, scientific references have probably reported the most severe cases.
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As other colleagues have suggested, BRAT1 should be added to the list of genes that are related to PE (11). Finally, we should insist on the necessity of genetic studies in
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patients with PE. Additional investigations will be worthwhile to determine the prevalence and contribution of these genes mutations to PE. Despite the clinical and etiology diversity of PE, genetic studies may be decisive for genetic counselling and
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management.
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ACCEPTED MANUSCRIPT REFERENCES
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1. Stromme P, Kanavin OJ, Abdelnoor M, et al. Incidence rates of progressive childhood encephalopathy in Oslo, Norway: a population based study. BMC pediatrics. 2007;7:25. PubMed PMID: 17597517. Pubmed Central PMCID: 1914055. 2. Surtees R. Understanding neurodegenerative disorders. Curr Paediatr. 2002;12(13):191-8. 3. Tomas Vila M, Vitoria Minana I, Gomez Gosalvez F, et al. [Epidemiology of progressive intellectual and neurological deterioration in childhood. A multicentre study in the Community of Valencia]. Anales de pediatria. 2013 May;78(5):303-7. PubMed PMID: 23063114. Epidemiologia del deterioro intelectual y neurologico progresivo en la infancia. Estudio multicentrico en la Comunidad Valenciana. 4. Nelson J, Smith M, Bittles AH. Consanguineous marriage and its clinical consequences in migrants to Australia. Clinical genetics. 1997 Sep;52(3):142-6. PubMed PMID: 9377802. 5. Weller M, Tanieri M, Pereira JC, et al. Consanguineous unions and the burden of disability: a population-based study in communities of Northeastern Brazil. American journal of human biology : the official journal of the Human Biology Council. 2012 Nov-Dec;24(6):835-40. PubMed PMID: 23042425. 6. Stromme P, Suren P, Kanavin OJ, et al. Parental consanguinity is associated with a seven-fold increased risk of progressive encephalopathy: a cohort study from Oslo, Norway. European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society. 2010 Mar;14(2):138-45. PubMed PMID: 19446480. 7. Stromme P, Magnus P, Kanavin OJ, et al. Mortality in childhood progressive encephalopathy from 1985 to 2004 in Oslo, Norway: a population-based study. Acta paediatrica. 2008 Jan;97(1):35-40. PubMed PMID: 18076719. 8. Puffenberger EG, Jinks RN, Sougnez C, et al. Genetic mapping and exome sequencing identify variants associated with five novel diseases. PloS one. 2012;7(1):e28936. PubMed PMID: 22279524. Pubmed Central PMCID: 3260153. 9. Saitsu H, Yamashita S, Tanaka Y, et al. Compound heterozygous BRAT1 mutations cause familial Ohtahara syndrome with hypertonia and microcephaly. Journal of human genetics. 2014 Dec;59(12):687-90. PubMed PMID: 25319849. 10. Saunders CJ, Miller NA, Soden SE, et al. Rapid whole-genome sequencing for genetic disease diagnosis in neonatal intensive care units. Science translational medicine. 2012 Oct 3;4(154):154ra35. PubMed PMID: 23035047. Pubmed Central PMCID: 4283791. 11. Straussberg R, Ganelin-Cohen E, Goldberg-Stern H, et al. Lethal neonatal rigidity and multifocal seizure syndrome--report of another family with a BRAT1 mutation. European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society. 2015 Mar;19(2):240-2. PubMed PMID: 25500575. 12. Exome Aggregation Consortium ExAC. ExAC Browser Beta 2015 [updated July]. Available from: http://exac.broadinstitute.org. 13. Aglipay JA, Martin SA, Tawara H, Lee SW, Ouchi T. ATM activation by ionizing radiation requires BRCA1-associated BAAT1. The Journal of biological chemistry. 2006 Apr 7;281(14):9710-8. PubMed PMID: 16452482. 14. Ouchi M, Ouchi T. Regulation of ATM/DNA-PKcs Phosphorylation by BRCA1-Associated BAAT1. Genes & cancer. 2010 Dec;1(12):1211-4. PubMed PMID: 21779444. Pubmed Central PMCID: 3092282. 15. So EY, Ouchi T. Functional interaction of BRCA1/ATM-associated BAAT1 with the DNAPK catalytic subunit. Experimental and therapeutic medicine. 2011 May;2(3):443-7. PubMed PMID: 22977523. Pubmed Central PMCID: 3440717.
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16. So EY, Ouchi T. BRAT1 deficiency causes increased glucose metabolism and mitochondrial malfunction. BMC cancer. 2014;14:548. PubMed PMID: 25070371. Pubmed Central PMCID: 4129107. 17. Khan S, Al Baradie R. Epileptic encephalopathies: an overview. Epilepsy research and treatment. 2012;2012:403592. PubMed PMID: 23213494. Pubmed Central PMCID: 3508533. 18. Willig LK, Petrikin JE, Smith LD, et al. Whole-genome sequencing for identification of Mendelian disorders in critically ill infants: a retrospective analysis of diagnostic and clinical findings. The Lancet Respiratory medicine. 2015 May;3(5):377-87. PubMed PMID: 25937001. Pubmed Central PMCID: 4479194.
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Figure 1.
Sleep video-EEG test, performed on a 25-month-old boy. Normal EEG results; no
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epileptic paroxysms were registered.
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Figures 2-3.
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On the left side, MRI study performed on a 19-month-old boy (2a-2b). 2a. Midsagittal T1-weighted image exhibits a severe vermian diffuse atrophy (arrow) with fourth ventricle enlargement. The corpus callosum is normal, and the brainstem
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has a normal size.
2b. Axial T2-weighted image shows enlarged interfolia spaces (arrow) and moderately
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reduced volume of both cerebellar hemispheres. On the right side, MRI study performed at 48 months old (3a-3b). 3a. Midsagittal T1-weighted image evidences a more severe vermian diffuse atrophy (arrow) with enlargement of the supravermian cistern and a wide fourth ventricle. The corpus callosum and the brainstem remain normal. 3b. Axial T2-weighted image shows severe symmetric atrophy of the cerebellar hemispheres (arrow).
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Table I. Clinical features, brain MRI findings and BRAT1 mutations. Puffenberg
Saunders CJ, et al.
Saitsu H
Straussbe
EG, et al.
(10)
et al. (9)
rg R, et
N=1
N=2
N=2
N=2
Clinical
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Features Dysmorphic
-
+
+
-
-
+ (at birth)
+ (at birth)
+
+
+
(postnatal
(postnatal
(postnatal
)
)
)
+
+
+
+
+
+
-
+
-
+
-
-
+
-
-
-
+
-
+
Rigidity/Hyper +
+
+
Dysautonomia
+
(apnea, bradycardia)
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Seizures
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tonia
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features Microcephaly
N=1
al. (11)
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(8)
Our case
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Brain MRI Cerebral
+ (mild
atrophy
hypoplasia of frontal lobes)
Cerebellar atrophy
-
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Before age
Age 5 months
4 months
Ages 1
Before
Alive at
year and
age 6
52
9 months
months
months
(1st case) and 3
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months (2nd case) Homozygo
mutation
us
Homozygous c.453_454insATCT
c.638_639i
TCTC
Homozyg
Compoun
d
ous
d
heterozyg ous
c.1173del G
heterozyg ous
c.176T>C
c.1564G>
and
A and
c.962_96
c.638dup
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nsA
Compoun
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BRAT1
3del
Amish
Mexican
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Population
Japanese Siblings
ArabMuslim Siblings
Spanish
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Acknowledgements: Part of this work was supported by R01CA79892, R01CA90631,
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and 5P30CA16056 from the National Institutes of Health, USA (T.O.).
ACCEPTED MANUSCRIPT Highlights • • •
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•
Homozygous and compound heterozygous BRAT1 mutations are related to severe progressive encephalopathy. The loss of BRAT1 expression may decrease neuronal proliferation and migration. We describe the oldest patient with BRAT1 mutations, progressive encephalopathy and without seizures. Whole exome sequencing may be useful for identifying the causes of progressive encephalopathies.